Glutamate or excitatory amino acid transporters (EAATs) are members of the Solute Carrier 1 family of transmembrane proteins. EAATs are key in the function of neuronal synapses responsible for the removal of excitatory neurotransmitters from the synaptic cleft after each neurotransmission. Malfunction of EAATs is involved in cerebral stroke, epilepsy, Alzheimer's disease, dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma. Thus, a profound understanding of the molecular determinants of EAAT function is crucial to understand these diseases. In the last decade, the structure and function of a prokaryotic glutamate transporter homolog, the sodium/aspartate symporter from archaebacterium Pyrococcus horikoshii, GltPh, has been extensively studied and made a perfect model system for EAAT studies. More recently a first structure of the human EAAT1 has been solved, but little is known about its transport dynamics and kinetics. In this project we will study both GltPh and hEAAT1 proteins, with the aim to characterize the so far elusive transport sub-states and kinetics in the ms and s range. While the transport kinetics of the prokaryotic GltPh are slow (seconds to tens of milliseconds), EAATs are expected to work at faster rates (~100 to ~1000 transport cycles per second). Here, we will reconstitute GltPh and hEAAT1 in lipid membranes and employ high-speed atomic force microscopy (HS-AFM) to directly image transport cycles of individual unlabeled glutamate transporters with the aim to resolve transport-related conformational sub-states and fast kinetics. To achieve this goal, in addition to HS-AFM (HS-AFM) imaging, we develop and employ HS-AFM line scanning (HS-AFM-LS) and HS-AFM height spectroscopy (HS-AFM-HS). In these two novel sub-modes, we reach millisecond and microsecond time resolution, respectively. This makes our approach unique in three ways: i) We analyze the dynamics of membrane transporters in membrane. ii) We analyze unlabeled single transporter molecules. iii) We reach so far inaccessible temporal resolution. These novel approaches allow to unveil the occluded state in GltPh (only the outward and inward facing states in the transport cycle were assigned to date), its lifetime, the frequency with which it is being visited, and if it is visited on passage of a complete cycle or if returns from the occluded state occur regularly. In addition, given the fast transport rates of the eukaryotic homologues, we will pioneer single molecule dynamics measurements on human EAAT1 and provide first insights into their transport state probabilities and kinetics. The overall goal of the project is to establish a new experimental tool to study millisecond and microsecond dynamics in transporters and to apply this tool towards uncovering intermediates states in the transport cycles and measure currently inaccessible fast transport kinetics on the single molecule level.